Patent Application
20110287467
The present invention is directed to methods of producing recombinant heme-binding proteins with complete heme incorporation and methods of using the same.
Heme proteins encompass a wide range of functions that include electron transfer, transport and storage of oxygen, production and sensing of nitric oxide, decomposition of reactive oxygen species, catalytic oxidation of substrates, signal transduction and control of gene expression (Gray et al., “Electron Transfer in Engineered Heme Enzymes,” Faseb. J. 11:A781-A781 (1997); Sono et al., “Heme-Containing Oxygenases,” Chem. Rev. 96:2841-2888 (1996); Alderton et al., “Nitric Oxide Synthases Structure, Function and Inhibition,” Biochem. J. 357:593-615 (2001); and Perutz et al., “A Haemoglobin that Acts as an Oxygen Sensor: Signalling Mechanism and Structural Basis of its Homology with PAS Domains,” Chem. Biol. 6:R291-R297 (1999)). Under incorporation of heme into recombinant proteins can adversely influence their characterization. Evaluation of enzymatic activity is systematically underestimated if a significant proportion of the recombinant sample does not contain heme. Furthermore, proteins devoid of heme may bind another non-native cofactor with a contaminating activity of its own. A pure protein sample is usually essential for structural characterization by techniques such as X-ray crystallography, as this method often requires a single, well-folded species for crystallization. Other methods of heme protein characterization are less sensitive to the level of heme incorporation, particularly if those methods directly detect the metallocofactor (e.g., UV/Vis, EPR, and Mossbauer spectroscopy) or rely on enzymatic activity. This is both good and bad in that under incorporation may not greatly affect the analysis, but heterogeneity in the sample may go undetected.
Incomplete heme incorporation into recombinant proteins has been a frequently encountered problem (Varadarajan et al., “Cloning, Expression in Escherichia-Coli, and Reconstitution of Human Myoglobin,” Pro. Nat. Acad. Sci. U.S.A. 82:5681-5684 (1985); Ishikawa et al., “Expression of Rat Heme Oxygenase in Escherichia-Coli as a Catalytically Active, Full-Length Form that Binds to Bacterial-Membranes,” Eur. J. Biochem. 202:161-165 (1991); Smith et al., “Expression of a Synthetic Gene for Horseradish Peroxidase-C in Escherichia-Coli and Folding and Activation of the Recombinant Enzyme with Ca-2+ and Heme,” J. Biol. Chem. 265:13335-13343 (1990); Kery et al., “Delta-Aminolevulinate Increases Heme Saturation and Yield of Human Cystathionine Beta-Synthase Expressed in Escherichia-Coli,” Archives Biochem. Biophys. 31:624-29 (1995); Varnado et al., “Properties of a Novel Periplasmic Catalase-Peroxidase from Escherichia Coli O157: H7,” Archives Biochem. Biophys. 42:1166-174 (2004); and Graves et al., “Enhancing Stability and Expression of Recombinant Human Hemoglobin in E-Coli: Progress in the Development of a Recombinant HBOC Source,” Biochimica Et Biophysica Acta-Proteins And Proteomics 1784:1471-1479 (2008)) and thus, techniques have been developed to improve heme loading (Kery et al., “Delta-Aminolevulinate Increases Heme Saturation and Yield of Human Cystathionine Beta-Synthase Expressed in Escherichia-Coli,” Archives Biochem. Biophys. 31:624′29 (1995); Varnado et al., “Properties of a Novel Periplasmic Catalase-Peroxidase from Escherichia Coli O157: H7,” Archives Biochem. Biophys. 42:1166-174 (2004); Graves et al., “Enhancing Stability and Expression of Recombinant Human Hemoglobin in E-Coli: Progress in the Development of a Recombinant HBOC Source,” Biochimica Et Biophysica Acta-Proteins And Proteomics 1784:1471-1479 (2008); Weickert et al., “Optimization of Heterologous Protein Production in Escherichia Coli,” Curr. Opinion In Biotechnol. 7:494-499 (1996); Shen et al., “Production of Unmodified Human Adult Hemoglobin in Escherichia-Coli,” Pro. Nat, Acad. Sci. U.S.A. 90:8108-8112 (1993); Varnado et al., “System for the Expression of Recombinant Hemoproteins in Escherichia Coli,” Prot. Exp. Pur. 35:76-83 (2004); and Vaniado et al., “Expression of Recombinant Hemoproteins in E. Coli Using a Heme Protein Expression System,” Biophys. J. 384A-384A (2007)). During induction of recombinant protein expression from highly active vectors, such as those that employ the T7-polymerase, a population of protein will fold without the heme co-factor under conditions where folding outpaces heme delivery (Weickert et al., “Blackmore, Stabilization of Apoglobin by Low Temperature Increases Yield of Soluble Recombinant Hemoglobin in Escherichia Coli,” App. Environ. Microbiol. 63:4313-4320 (1997)). Supplementing the growth media with δ-amino levulinic acid (d-ALA), a precursor in the C5 heme biosynthesis pathway, increases levels of heme biosynthesis and thereby heme incorporation into the target protein (Kery et al., “Delta-Aminolevulinate Increases Heme Saturation and Yield of Human Cystathionine Beta-Synthase Expressed in Escherichia-Coli,” Archives Biochem. Biophys. 31:624-29 (1995); Pcsce ct al., “The 109 Residue Nerve Tissue Minihemoglobin from Cerebratulus Lacteus Highlights Striking Structural Plasticity of the Alpha-Helical Globin Fold,” Structure 10:725-735 (2002); and Summerford et al., “Bacterial Expression of Scapharca Dimeric Hemoglobin—A Simple-Model System for Investigating Protein Cooperativity,” Prot. Engineer. 8:593-599 (1995)). Increased heme biosynthesis rates through d-ALA supplementation does not achieve complete heme incorporation into all heme-binding proteins (Weickert et al., “Optimization of Heterologous Protein Production in Escherichia Coli,” Curr. Opinion In Biotechnol. 7:494-499 (1996); Shen et al., “Production of Unmodified Human Adult Hemoglobin in Escherichia-Coli,” Pro. Nat. Acad. Sci. U.S.A. 90:8108-8112 (1993); Weickert et al., “High-Fidelity Translation of Recombinant Human Hemoglobin in Escherichia Coli,” Appl. Environ. Microbiol. 64:589-1593 (1998); and Wcickert et al., “A Mutation that Improves Soluble Recombinant Hemoglobin Accumulation in Escherichia Coli in Heme Excess,” App. Environ. Microbiol. 65:640-647 (1999)).
Another technique for increasing home incorporation into recombinant proteins involves supplying the bacteria with hemin in the growth media. However, most E. coli strains do not possess an efficient heme transport system, and thus uptake of hemin relies on diffusion through the cell membrane. As a result, hemin feeding is much more effective with strains that co-express heme transport genes from other gram-negative bacteria, along with the heme-protein of interest (Graves et al., “Enhancing Stability and Expression of Recombinant Human Hemoglobin in E-Coli: Progress in the Development of a Recombinant HBOC Source,” Biochimica Et Biophysica Acta—Proteins And Proteomics 1784:1471-1479 (2008); Varnado et al., “System for the Expression of Recombinant Hemoproteins in Escherichia Coli,” Prot. Exp. Pur. 35:76-83 (2004); and Varnado et al., “Expression of Recombinant Hemoproteins in E. Coli Using a Heme Protein Expression System,” Biophys. J. 384A-384A (2007)). For example, co-expression of the heme transport system from P. shigelloides, which consists of the proteins Hug A/B/C/D, TonB, and Exb B/D, while also supplementing the growth media with hemin, results in higher amounts of the target holo-protein (in this case hemoglobin) (Graves et al., “Enhancing Stability and Expression of Recombinant Human Hemoglobin in E-Coli: Progress in the Development of a Recombinant HBOC Source,” Biochimica Et Biophysica Acta—Proteins And Proteomics 1784:1471-1479 (2008)). A similar method involves the co-expression of the heme receptor ChuA from E. coli. strain O157:H7 to enhance hemin (Varnado et al., “System for the Expression of Recombinant Hemoproteins in Escherichia Coli,” Prot. Exp. Pur. 35:76-83 (2004)). This latter method also shows a significant increase in the amount of heme-loaded protein generated, although in both cases, the ratio of holoprotein:apoprotein was not evaluated. Another approach utilizes the heme-permeability of E. coli strain RP523, which has the hem B, porphobilinogen synthase gene disrupted to prevent native heme synthesis. All heme and/or heme analogs are procured by the cells from the growth media and incorporation is nearly stoichiometric for proteins expressed in the cytoplasm (0.8-1.0 heme/heme analog per protein) (Woodward et al., “An Escherichia Coli Expression-Based Method for Heme Substitution,” Nat. Methods 4:43-45 (2007)).
Full incorporation of heme in recombinant proteins is also important for commercial applications. For example, the feasibility of employing recombinant human hemoglobin as an oxygen delivery pharmaceutical is limited by the yield of holoprotein that can be made in E. coli (Graves et al. “Enhancing Stability and Expression of Recombinant Human Hemoglobin in E-Coli: Progress in the Development of a Recombinant HBOC Source,” Biochimica Et Biophysica Acta—Proteins And Proteomics 1784:1471-1479 (2008)). Some of the methods discussed above, while effective, require co-expression of several heme transport proteins, which could limit yields, and/or require addition of the heme cofactor.
The present invention is directed to overcoming these and other deficiencies in the art.
A first aspect of the present invention relates to a method of producing a functional recombinant heme-binding protein. This method involves co-expressing a recombinant heme-binding protein and recombinant ferrochelatase protein, or polypeptide thereof, under conditions effective for complete heme incorporation into the recombinantly produced heme-binding protein, thereby producing a functional heme-binding protein.
A second aspect of the present invention relates to a system for producing functional heme-binding proteins. This system comprises an expression system and one or more expression constructs encoding a recombinant heme-binding protein and a recombinant ferrochelatase.
A third aspect of the present invention relates to a purified preparation of recombinant functional heme-binding protein.
A fourth aspect of the present invention relates to a method of identifying an agent that modulates activity of a heme-binding protein. This method involves providing a candidate agent and providing a recombinant functional heme-binding protein. This method further involves contacting the candidate agent with the recombinant functional heme-binding protein under conditions at which the functional heme-binding protein is active and comparing the activity of the functional heme-binding protein as a result of said contacting to the activity of the heme-binding protein alone, both under said conditions at which the heme-binding protein is active. A candidate agent that modulates the activity of a heme-binding protein is identified based on said comparing.
Another aspect of the present invention relates to a method of evaluating the metabolism of an agent by a heme-binding protein. This method involves providing a candidate agent and providing a recombinant functional heme-binding protein. This method further involves contacting the candidate agent with the recombinant functional home-binding protein under conditions at which the functional heme-binding protein is active and comparing the activity of the functional heme-binding protein as a result of said contacting to the activity of the heme-binding protein alone, both under said conditions at which the heme-binding protein is active. The metabolism of the candidate agent by a heme-binding protein is evaluated based on said comparing.
The present invention is a straightforward and inexpensive method for high fidelity incorporation of heme into recombinantly overexpressed heme proteins. Co-expression of just one native protein, ferrochelatase (FC), in the presence of δ-ALA is sufficient to achieve 100% heme incorporation into three unrelated home proteins derived from different organisms. Since pre-existing methods of recombinant heme-binding protein production result in sub-optimal heme incorporation and varying amounts of protein production depending on the protein of interest, the ability to achieve complete heme incorporation as described herein, has important implications for heme-binding protein biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial heme-binding proteins with high activity.
A first aspect of the present invention relates to a method of producing functional recombinant heme-binding proteins. This method involves co-expressing a recombinant heme-binding protein and recombinant ferrochelatase protein, or polypeptide thereof, under conditions effective for complete heme incorporation into the recombinantly produced heme-binding protein, thereby producing a functional heme-binding protein.
In accordance with this aspect of the invention, a heme-binding protein encompasses any protein that contains a heme prosthetic group either covalently or noncovalently bound to itself. Heme-binding proteins have diverse biological functions including, oxygen transport, catalysis, active membrane transport, electron transport, and sensory. The various classes of heme-binding proteins that are encompassed by the methods of the present invention include, without limitation, globins (e.g., hemoglobin, myoglobin, neuroglobin, cytoglobin, leghemoglobin), cytochromes (e.g., a-, b-, and c-types, cd1-nitrite reductase, cytochrome oxidase), transferrins (e.g., lactotransferrin, serotransferrin, melanotransferrin), bacterioferririns, hydroxylamine oxidoreductase, nitrophorins, peroxidases (e.g., lignin peroxidase), cyclooxygenases (e.g., COX-1, COX-2, COX-3, prostaglandin H synthase), catalases, cytochrome P-450s, chloroperoxidases, PAS-domain heme sensors, H-NOX heme sensors (e.g., soluble guanylate cyclase, FixL, DOS, HemAT, and CooA), heme-oxygenases, and nitric oxide synthases. The recombinant heme-binding protein produced using the methods of the present invention can be prokaryotic or eukaryotic. For example, in one embodiment of the present invention the recombinant heme-binding protein is mammalian, preferably human. In another embodiment of the present invention the recombinant heme-binding protein is bacterial. In yet another embodiment of the present invention, the recombinant heme-binding protein is fungal, preferably yeast.
Table 1 provides a non-exhaustive list of exemplary human heme-binding proteins that are suitable for production using the methods of the present invention. Table 1 identifies each heme-binding protein by its Universal Protein Resource Knowledgebase (UniProtKB)/Swiss Prot accession number, which provides the amino acid sequence of the identified protein, and the EMBL Nucleotide Database accession number, which provides the nucleotide sequence encoding the heme-binding protein. The UniProtKB/Swiss Prot and EMBL accession numbers, along with the corresponding amino acid and nucleotide sequence information for each entry in Table 1 is hereby incorporated by reference. Table 1 further identifies the UniProtKB/Swiss Prot entry name, protein names and gene names for each identified heme-binding protein.
Table 2 provides a list of lignin peroxidases, which are heme-binding proteins also suitable for production using the methods of the present invention. As described herein, lignin peroxidases metabolism the lignin of plant cell walls which facilitates the breakdown of cell wall polysaccharides to simple sugars and the subsequent conversion of these sugars to usable bio-fuel. Table 2 identifies each lignin peroxidase by its UniProtKB/Swiss Prot accession number, which provides the amino acid sequence of the protein, and the EMBL Nucleotide Database accession number, which provides the encoding nucleotide sequence. The UniProtKB/Swiss Prot and EMBL accession numbers, along with the corresponding amino acid and nucleotide sequence information for each entry in Table 2 is hereby incorporated by reference. Table 2 further identifies the UniProtKB/Swiss Prot entry name, protein names, protein family, gene names, and organism for each entry.
Traditional methods of producing recombinant heme-binding proteins generate recombinant proteins having incomplete heme incorporation. Under incorporation of heme into recombinant proteins can adversely influence their function. In contrast to these traditional methods, the methods of the present invention generate recombinant heme-binding proteins that have complete heme incorporation. Therefore, these proteins are completely functional and are more suitable for research, clinical, and commercial applications. Incomplete heme incorporation into a recombinant protein can be detected by the presence of free base porphyrin using fluorescence spectroscope
indicates data missing or illegible when filed
Arthromyces ramosus
Coprinopsis cinerea
aspergillata)
Coprinopsis cinerea
aspergillata)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phanerochaete
chrysosporium (White-
pruinosum)
Phlebia radiata (White-
Phlebia radiata (White-
Phlebia radiata (White-
Pleurotus eryngii
Pleurotus eryngii
Pleurotus eryngii
Taiwanofungus
camphoratus (Poroid
Trametes versicolor
Trametes versicolor
Trametes versicolor
(excitation at 397 nm) and resonance Raman spectroscopy. As demonstrated herein, recombinant heme-binding proteins generated using the methods of the present invention show no evidence of free base porphryin incorporation.
The recombinant ferrochelatase protein or polypeptide used in the methods of the present invention includes any recombinant ferrochelatase, or polypeptide thereof that is capable of catalyzing the insertion of ferrous iron into protoporphyrin IX to form protoheme (i.e., enzyme classification (EC) no. 4.99.1.1). Well over 400 ferrochelatases from both prokaryotes and eukaryotes have been characterized and are known in the art (see e.g., UniProtKB, (GenBank). Table 3 below provides a listing of 429 known ferrochelatase enzymes from the UniProtKB database identified by UniProtKB/Swiss-Pro Accession number, protein name, gene name, organism, EC number, and EMBL accession number. Each of the ferrochelatases listed in Table 3 is suitable for use in the present invention. The UniProtKB/Swiss Prot and EMBL accession numbers, along with the corresponding amino acid and nucleotide sequence information for each entry in Table 3 is hereby incorporated by reference. Selection of an appropriate ferrochelatase to use when carrying out the methods of the present invention is based on the recombinant heme-binding protein being produced and/or the chosen expression system.
It is to be understood that the present invention contemplates the use of any bacterial, archaeal, fungal, plant, and animal ferrochelatase known in the art. The present invention also contemplates the use of polypeptide fragments of ferrochelatase which retain iron insertion activity (i.e., suitable proteins or polypeptides have an enzyme classification of 4.99.1.1). Methods of analyzing and measuring the enzyme activity of ferrochelatase are well known in the art (see e.g., Miyamoto et al., “Overproduction, Purification, and Characterization of Ferrochelatase from Escherichia coli,” J. Biochem. 115:545-551 (1994) and Taketani et al., “Rat Liver Ferrochelatase,” J. Biol. Chem., 256(24):12748-53 (1981), which are hereby incorporated by reference in their entirety). Therefore, suitable ferrochelatase proteins or polypeptides not identified in Table 3 can be readily identified by one of skill in the art using these enzyme activity assays.
Arabidopsis thaliana (Mouse-ear
Bacillus anthracis
Bacillus cereus (strain ATCC 10987)
Bacillus cereus (strain ATCC 14579/
Bacillus cereus (strain ZK/E33L)
Bacillus thuringiensis subsp.
konkukian
Oryza sativa subsp. japonica (Rice)
Shewanella oneidensis
Arabidopsis thaliana (Mouse-ear
Bacillus anthracis
Bacillus cereus (strain ATCC 10987)
Bacillus cereus (strain ATCC 14579/
Bacillus cereus (strain ZK/E33L)
Bacillus thuringiensis subsp.
konkukian
Oryza sativa subsp. japonica (Rice)
Shewanella oneidensis
Acinetobacter sp. (strain ADP1)
Acinetobacter baumannii (strain
Acinetobacter baumannii (strain
Acinetobacter baumannii (strain
Acinetobacter baumannii (strain SDF)
Acinetobacter baumannii (strain ATCC
Acinetobacter baumannii (strain AYE)
Acidobacterium capsulatum (strain
Actinobacillus pleuropneumoniae
Actinobacillus pleuropneumoniae
Actinobacillus pleuropneumoniae
Aeromonas hydrophila subsp.
hydrophila (strain ATCC 7966/NCIB
Agrobacterium tumefaciens (strain
Agrobacterium vitis (strain S4/ATCC
Alcanivorax borkumensis (strain SK2/
Alilvibrio salmonicida (strain LFI1238)
Alteromonas macieodii (strain DSM
Anabaena variabilis (strain ATCC
Anoxybacillus flavithermus (strain DSM
Aquifex aeolicus
Aromatoleum aromaticum (strain
Azoarcus sp. (strain BH72)
Azotobacter vinelandii (strain DJ/
Bacillus halodurans
Bacillus pumilus (strain SAFR-032)
Bacillus clausii (strain KSM-K16)
Bacillus subtilis
Bacillus weihenstephanensis (strain
Bdellovibrio bacteriovorus
Bordetella bronchiseptica (Alcaligenes
bronchisepticus)
Bordetella parapertussis
Bordetella pertussis
Bos taurus (Bovine)
Bradyrhizobium japonicum
Bradyrhizobium sp. (strain BTAi1/
Brucella abortus (strain S19)
Brucella abortus (strain 2308)
Brucella abortus
Brucella canis (strain ATCC 23365/
Brucella melitensis biotype 2 (strain
Brucella melitensis
Brucella ovis (strain ATCC 25840/
Brucella suis (strain ATCC 23445/
Brucella suis
Burkholderia ambifaria (strain MC40-6)
Burkholderia cenocepacia (strain AU
Burkholderia cenocepacia (strain MC0-
Burkholderia cenocepacia (strain
Burkholderia ambifaria (strain ATCC
cepacia (strain AMMD))
Burkholderia mallei (strain NCTC
Burkholderia mallei (strain NCTC
Burkholderia mallei (Pseudomonas
mallei)
Burkholderia mallei (strain SAVP1)
Burkholderia pseudomallei (strain
Burkholderia pseudomallei (strain
Burkholderia pseudomallei (strain 668)
Burkholderia phymatum (strain DSM
Burkholderia phytofirmans (strain DSM
Burkholderia pseudomallei
Burkholderia sp. (strain 383)
Burkholderia thailandensis (strain
Burkholderia vietnamiensis (strain G4/
Burkholderia xenovorans (strain
Campylobacter concisus (strain 13826)
Campylobacter fetus subsp. fetus
Campylobacter jejuni subsp. jejuni
Campylobacter jejuni subsp. doylei
Campylobacter jejuni
Campylobacter jejuni subsp. jejuni
Campylobacter jejuni (strain RM1221)
Caulobacter crescentus (strain
Caulobacter crescentus (Caulobacter
vibrioides)
Gallus gallus (Chicken)
Chlamydophila abortus
Chlamydophila caviae
Chlamydophila felis (strain Fe/C-56)
Chlamydia muridarum
Chlamydia pneumoniae
Chlamydia trachomatis serovar L2
Chlamydia trachomatis serovar A
Chlamydia trachomatis serovar L2b
Chlamydia trachomatis
Chromobacterium violaceum
Corynebacterium diphtheriae
Corynebacterium efficiens
Corynebacterium glutamicum (strain
Corynebacterium glutamicum
Coxiella bumetii (strain CbuK_Q154)
Coxiella bumetii (strain CbuG_Q212)
Coxiella bumetii (strain Dugway 5J108-
Coxiella burnetii
Cucumis sativus (Cucumber)
Cupriavidus necator (strain ATCC
Cupriavidus pinatubonensis (strain
eutrophus) (Ralstonia eutropha)
Cupriavidus taiwanensis (strain R1/
Cyanothece sp. (strain PCC 7425/
Cyanothece sp. (strain PCC 7424)
Cyanothece sp. (strain PCC 8801)
Dechloromonas aromatica (strain
Deinococcus radiodurans
Desulfotalea psychrophila
Dictyostelium discoideum (Slime mold)
Drosophila melanogaster (Fruit fly)
Escherichia coli O139:H28 (strain
Escherichia coli O127:H6 (strain
Escherichia coil O45:K1 (strain S88/
Escherichia coli (strain 55989/EAEC)
Escherichia coli O157:H7
Escherichia coli O157:H7 (strain
Escherichia coli O7:K1 (strain IAI39/
Escherichia coli O81 (strain ED1a)
Escherichia coli O8 (strain IAI1)
Escherichia coli (strain K12/MC4100/
Escherichia coli (strain K12/DH10B)
Escherichia coli O9:H4 (strain HS)
Escherichia coli O1:K1/APEC
Escherichia coli O6:K15:H31 (strain
Escherichia coli O6
Escherichia coli (strain ATCC 8739/
Escherichia coli (strain K12)
Escherichia coli O17:K52:H18 (strain
Escherichia coli (strain SE11)
Escherichia coli (strain SMS-3-5/
Escherichia coli (strain UTI89/UPEC)
Edwardsiella ictaluri (strain 93-146)
Enterobacter sp. (strain 638)
Enterococcus faecalis (Streptococcus
faecalis)
Enterobacter sakazakii (strain ATCC
Erwinia carotovora subsp. atroseptica
Erwinia tasmaniensis (strain DSM
Escherichia fergusonii (strain ATCC
Frankia alni (strain ACN14a)
Francisella tularensis subsp. tularensis
Francisella tularensis subsp. holarctica
Francisella tularensis subsp. holarctica
Francisella tularensis subsp.
mediasiatica (strain FSC147)
Francisella tularensis subsp. novicida
Francisella tularensis subsp. holarctica
Francisella tularensis subsp. tularensis
Francisella tularensis subsp. tularensis
Geobacter bemidjiensis (strain Bem/
Geobacillus kaustophilus
Geobacter metallireducens (strain GS-
Geobacter sp. (strain FRC-32)
Geobacter sulfurreducens
Geobacter sp. (strain M21)
Geobacillus sp. (strain WCH70)
Geobacillus thermodenitrificans (strain
Geobacter uraniireducens (strain Rf4)
Gloeobacter violaceus
Gluconobacter oxydans
Haemophilus influenzae (strain 86-
Haemophilus influenzae (strain PittEE)
Haemophilus influenzae (strain ATCC
Hamiltonella defensa subsp.
Acyrthosiphon pisum (strain 5AT)
Helicobacter acinonychis (strain
Helicobacter hepaticus
Helicobacter pylori (strain P12)
Helicobacter pylori (strain G27)
Helicobacter pylori (strain HPAG1)
Helicobacter pylori J99
Helicobacter pylori (strain Shi470)
Helicobacter pylori (Campylobacter
pylori)
Herpetosiphon aurantiacus (strain
Herminiimonas arsenicoxydans
Hordeum vulgare (Barley)
Homo sapiens (Human)
Hyphomonas neptunium (strain ATCC
Idiomarina loihiensis
Janthinobacterium sp. (strain
Klebsiella pneumoniae (strain 342)
Lactobacillus casei (strain ATCC 334)
Lactobacillus casei (strain BL23)
Lactococcus lactis subsp. lactis
Lactococcus lactis subsp. cremoris
Lactococcus lactis subsp. cremoris
Lactobacillus plantarum
Lactobacillus reuteri (strain DSM
Lactobacillus reuteri (strain JCM 1112)
Legionella pneumophila (strain Paris)
Legionella pneumophila (strain Corby)
Legionella pneumophila subsp.
pneumophila (strain Philadelphia 1/
Legionella pneumophila (strain Lens)
Leifsonia xyli subsp. xyli
Leptospira biflexa serovar Patoc (strain
Leptospira biflexa
Leptospira borgpetersenii serovar
Leptospira borgpetersenii serovar
Leptospira biflexa serovar Patoc (strain
Leuconostoc mesenteroides subsp.
mesenteroides (strain ATCC 8293/
Listeria innocua
Listeria monocytogenes serotype 4b
Listeria monocytogenes
Mannheimia succiniciproducens (strain
Marinobacter aquaeolei (strain ATCC
Marinomonas sp. (strain MWYL1)
Mesorhizobium sp. (strain BNC1)
Methylococcus capsulatus
Methylobacillus flagellatus (strain KT/
Methylibium petroleiphilum (strain
Methylocella silvestris (strain BL2/
Microcystis aeruginosa (strain NIES-
Mus musculus (Mouse)
Mycobacterium avium (strain 104)
Mycobacterium abscessus (strain
Mycobacterium avium
Mycobacterium bovis
Mycobacterium bovis (strain BCG/
Mycobacterium bovis (strain BCG/
Mycobacterium gilvum (strain PYR-
Mycobacterium leprae (strain Br4923)
Mycobacterium leprae
Mycobacterium marinum (strain ATCC
Mycobacterium paratuberculosis
Mycobacterium smegmatis (strain
Mycobacterium sp. (strain JLS)
Mycobacterium sp. (strain KMS)
Mycobacterium sp. (strain MCS)
Mycobacterium tuberculosis (strain
Mycobacterium tuberculosis
Mycobacterium ulcerans (strain Agy99)
Mycobacterium vanbaalenii (strain
Neisseria gonorrhoeae (strain ATCC
Neisseria meningitidis serogroup A
Neisseria meningitidis serogroup B
Neisseria meningitidis serogroup C/
Nitrosomonas europaea
Nitrobacter hamburgensis (strain X14/
Nitrosococcus oceani (strain ATCC
Nitratiruptor sp. (strain SB155-2)
Nocardia farcinica
Nostoc punctiforme (strain ATCC
Nostoc sp. (strain PCC 7120/UTEX
Oceanobacillus iheyensis
Ochrobactrum anthropi (strain ATCC
Opitutus terrae (strain DSM 11246/
Oryza sativa subsp. indica (Rice)
Pan troglodytes (Chimpanzee)
Protochlamydia amoebophila (strain
Pasteurella multocida
Pelobacter propionicus (strain DSM
Pelagibacter ubique
Phenylobacterium zucineum (strain
Photorhabdus luminescens subsp.
laumondii
Photobacterium profundum
Polaromonas sp. (strain JS666/ATCC
Porphyromonas gingivalis (strain
Porphyromonas gingivalis
Propionibacterium freudenreichii
Prochlorococcus marinus (strain MIT
Prochlorococcus marinus (strain
Prochlorococcus marinus (strain MIT
Prochlorococcus marinus (strain MIT
Prochlorococcus marinus (strain MIT
Prochlorococcus marinus (strain MIT
Prochlorococcus marinus (strain MIT
Prochlorococcus marinus
Proteus mirabilis (strain HI4320)
Prochlorococcus marinus (strain MIT
Prochlorococcus marinus subsp.
pastoris (strain CCMP1986/MED4)
Prochlorococcus marinus (strain
Prochlorococcus marinus (strain
Pseudomonas syringae pv.
Pseudoalteromonas atlantica (strain
Pseudomonas aeruginosa (strain PA7)
Pseudomonas aeruginosa (strain
Pseudomonas aeruginosa (strain
Pseudomonas aeruginosa
Pseudomonas entomophila (strain
Pseudomonas fluorescens (strain Pf-5/
Pseudomonas fluorescens biotype C
Pseudomonas fluorescens (strain
Pseudomonas mendocina (strain ymp)
Pseudomonas putida (strain F1/
Pseudomonas fluorescens (strain Pf0-
Pseudomonas putida (strain GB-1)
Pseudomonas putida (strain KT2440)
Pseudomonas putida (strain W619)
Pseudomonas syringae pv. tomato
Pseudomonas syringae pv. syringae
Pseudomonas stutzeri (strain A1501)
Psychrobacter arcticus (strain DSM
Psychrobacter cryohalolentis (strain
Psychrobacter sp. (strain PRwf-1)
Ralstonia metallidurans (strain CH34/
Ralstonia pickettii (strain 12J)
Ralstonia solanacearum
Rhizobium etli (strain CIAT 652)
Rhizobium etli (strain CFN 42/ATCC
Rhizobium leguminosarum bv. viciae
Rhizobium loti (Mesorhizobium loti)
Rhizobium leguminosarum bv. trifolii
Rhizobium meliloti (Sinorhizobium
meliloti)
Rhodopirellula baltica
Rhodobacter capsulatus
Rhodopseudomonas palustris (strain
Rhodopseudomonas palustris (strain
Rhodopseudomonas palustris
Rhodopseudomonas palustris (strain
Rhodopseudomonas palustris (strain
Rickettsia bellii (strain OSU 85-389)
Rickettsia bellii (strain RML369-C)
Rickettsia canadensis (strain McKiel)
Rickettsia conorii
Rickettsia felis (Rickettsia azadi)
Rickettsia prowazekii
Rickettsia typhi
Salmonella agona (strain SL483)
Salmonella arizonae (strain ATCC
Salmonella choleraesuis
Salmonella dublin (strain
Salmonella enteritidis PT4 (strain
Salmonella gallinarum (strain 287/91/
Salmonella heidelberg (strain SL476)
Salmonella newport (strain SL254)
Salmonella paratyphi A
Salmonella paratyphi B (strain ATCC
Salmonella paratyphi C (strain
Salmonella paratyphi A (strain
Salmonella schwarzengrund (strain
Salmonella typhi
Salmonella typhimurium
Schizosaccharomyces pombe (strain
Serratia proteamaculans (strain 568)
Shewanella denitrificans (strain OS217/
Shigella boydii serotype 18 (strain
Shigella boydii serotype 4 (strain
Shigella dysenteriae serotype 1 (strain
Shigella flexneri serotype 5b (strain
Shigella flexneri
Shigella sonnei (strain Ss046)
Sodalis glossinidius (strain morsitans)
Staphylococcus aureus (strain COL)
Staphylococcus aureus (strain Mu50/
Staphylococcus aureus (strain N315)
Staphylococcus aureus (strain
Staphylococcus aureus (strain
Staphylococcus aureus (strain MW2)
Staphylococcus epidermidis (strain
Staphylococcus epidermidis (strain
Staphylococcus saprophyticus subsp.
saprophyticus (strain ATCC 15305/
Streptomyces avermitilis
Streptomyces coelicolor
Streptomyces griseus subsp. griseus
Stenotrophomonas maltophilia (strain
Streptococcus mutans
Streptococcus pneumoniae serotype 2
Streptococcus pneumoniae (strain
Streptococcus pneumoniae (strain
Streptococcus pneumoniae (strain
Streptococcus pneumoniae
Streptococcus pneumoniae (strain
Streptococcus pneumoniae (strain
Streptococcus pneumoniae (strain
Streptococcus pneumoniae (strain
Sulfurimonas denitrificans (strain
Sulfurihydrogenibium sp. (strain
Symbiobacterium thermophilum
Synechococcus elongatus (strain PCC
Synechococcus sp. (strain JA-3-3Ab)
Synechococcus sp. (strain JA-2-3B′a(2-
Synechococcus sp. (strain ATCC
Synechococcus sp. (strain ATCC
Synechococcus sp. (strain WH7803)
Synechococcus sp. (strain WH8102)
Synechococcus sp. (strain RCC307)
Synechococcus sp. (strain CC9311)
Synechococcus sp. (strain CC9605)
Synechocystis sp. (strain ATCC 27184/
Teredinibacter tumerae (strain ATCC
Thermoplasma acidophilum (strain
Thermosynechococcus elongatus
Thermus thermophilus (strain HB27/
Thermoplasma volcanium (strain
Thiomicrospira crunogena (strain XCL-
Thiobacillus denitrificans (strain ATCC
Thioalkalivibrio sp. (strain HL-EbGR7)
Trichodesmium erythraeum (strain
Tropheryma whipplei (strain TW08/27)
Tropheryma whipplei (strain Twist)
Vibrio cholerae
Vibrio fischeri (strain ATCC 700601/
Vibrio fischeri (strain MJ11)
Vibrio parahaemolyticus
Vibrio splendidus (strain LGP32)
Vibrio vulnificus
Vibrio vulnificus (strain YJ016)
Wigglesworthia glossinidia brevipalpis
Wolbachia pipientis wMel
Wolbachia pipientis subsp. Culex
pipiens (strain wPip)
Wolinella succinogenes
Wolbachia sp. subsp. Brugia malayi
Wolbachia sp. subsp. Drosophila
simulans (strain wRi)
Xanthomonas axonopodis pv. citri
Xanthomonas campestris pv.
campestris
Xenopus laevis (African clawed frog)
Xylella fastidiosa (strain M23)
Xylella fastidiosa
Xylella fastidiosa (strain M12)
Xylella fastidiosa (strain Temecula1/
Saccharomyces cerevisiae (strain
Yersinia enterocolitica serotype O:8/
Yersinia enterocolitica
Yersinia pseudotuberculosis serotype
Yersinia pestis bv. Antiqua (strain
Yersinia pseudotuberculosis serotype
Yersinia pestis
Yersinia pestis bv. Antiqua (strain
Yersinia pestis bv. Antiqua (strain
Yersinia pestis (strain Pestoides F)
Yersinia pseudotuberculosis
Yersinia pseudotuberculosis serotype
Zymomonas mobilis
Suitable ferrochelatases of the present invention can also be identified by homology or sequence identity to the ferrochelatases disclosed in Table 3. Suitable ferrochelatases include those having an amino acid or nucleotide sequence identity of at least about 25 percent, more preferably at least about 30 to 40 percent, more preferably at least 50 to 60 percent, more preferably at least about 70 to 80 percent, most preferably at least about 85 to 95 percent as compared to a characterized ferrochelatase sequence of Table 3.
Mutant or modified ferrochelatase enzymes that retain their enzymatic activity (i.e., capable of catalyzing the insertion of ferrous iron into protoporphyrin IX to form protoheme (EC 4.99.1.1)) are also suitable for use in the present invention. Modifications or mutations to known ferrochelatase nucleotide sequences, such as nucleotide deletions, insertions, or substitutions that do not alter the coding sequence of the active region of the ferrochelatase enzyme or do not alter the activity of the encoded ferrochelatase enzyme are suitable for use in the methods of the present invention. For example, eukaryotic ferrochelatases possess an amino-terminus signal sequence that is lacking in prokaryote ferrochelatases (Dailey et al., “Ferrochelatase at the Millennium: Structures, Mechanisms, and [2Fe-2S] Clusters,” Cell. Mol. Life. Sci. 57:1909-1926 (2000) which is hereby incorporated by reference in its entirety). Therefore, recombinant eukaryotic ferrochelatase sequences containing modifications or deletions to this portion of the encoded protein that do not alter enzyme activity are suitable for use in the present invention. Likewise, eukaryotic ferrochelatases also possess a carboxyl-terminal extension sequence that is lacking in a majority of prokaryotic ferrochelatases (Dailey et al., “Ferrochelatase at the Millennium: Structures, Mechanisms, and [2Fe-2S] Clusters,” Cell. Mol. Life. Sci. 57:1909-1926 (2000), which is hereby incorporated by reference in its entirety). Recombinant eukaryotic ferrochelatase sequences containing modifications or deletions to this extension region are also suitable for use in the methods of the present invention. Similarly, recombinant ferrochelatase sequences containing single-base mutations that do not alter the conserved amino acid residues of the ferrochelatase protein acids (Dailey et al., “Ferrochelatase at the Millennium: Structures, Mechanisms, and [2Fe-2S] Clusters,” Cell. Mol. Life. Sci. 57:1909-1926 (2000), which is hereby incorporated by reference in its entirety) or the enzyme active site are also suitable for use in the present invention.
In accordance with this aspect of the invention, co-expression of the recombinant heme-binding protein and ferrochelatase protein, or polypeptide thereof, is carried out in the presence of one or more heme precursors, Suitable heme precursors include, without limitation, δ-amino levulinic acid, succinyl CoA, glycine, glutamate, glutamate-1 semialdehyde, porphobilinogen, hydroxymethylbilane, and protoporphyrin.
Co-expression of the recombinant heme-binding protein and ferrochelatase protein or polypeptide can be carried out in any one of the commonly known systems that are available in the art for heterologous protein expression, including, without limitation, eukaryotic and prokaryotic expression systems, and cell-free translation systems as described herein.
Techniques and protocols for manipulation of nucleic acids, including, for example, the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds., (1992), which is hereby incorporated by reference in its entirety.
Typically, a nucleic acid molecule encoding all or part of a protein of interest, i.e., a heme-binding protein, is obtained using methods such as those described herein. The protein-encoding nucleic acid sequence is cloned into an expression vector that is suitable for the particular host cell of interest using standard recombinant DNA procedures. Suitable expression vectors include those which contain replicon and control sequences that are derived from species compatible with the host cell.
Expression vectors include (among other elements) regulatory sequences (e.g., promoters) that can be operably linked to the desired protein-encoding nucleic acid molecule to cause the expression of such nucleic acid molecule in the host cell. Together, the regulatory sequences and the protein-encoding nucleic acid sequence are an expression construct. Expression vectors may also include an origin of replication, marker genes that provide phenotypic selection in transformed cells, one or more other promoters, and a polylinker region containing several restriction sites for insertion of heterologous nucleic acid sequences.
Expression vectors useful for expression of heterologous protein(s) in a multitude of host cells are well known in the art (Sambrook and Russell, Molecular Cloning: a Laboratory Manual 3rd ed. (2001), which is hereby incorporated by reference in its entirety), and some specific examples are provided herein. The host cell is transfected with (or infected with a virus containing) the expression vector using any method suitable for the particular host cell. Such transfection methods are also well known in the art and non-limiting exemplary methods are described herein. The transfected (also called, transformed) host cell is capable of expressing the protein encoded by the corresponding nucleic acid sequence in the expression construct. Transient or stable transfection of the host cell with one or more expression vectors is contemplated by the present disclosure.
In one embodiment of the present invention, the nucleotide sequence encoding the desired heme-binding protein is inserted into one expression vector and the nucleotide sequence encoding the ferrochelatase is inserted into a second expression vector. In this embodiment, the two expression vectors are co-transfected into an appropriate host cell for transcription and translation. In another embodiment of the invention, both nucleotide sequences are inserted into one expression vector and the single expression vector encodes both the recombinant heme-binding protein and the recombinant ferrochelatase.
Many different types of cells may be used to express heterologous proteins, such as bacterial, archaeal, yeast, fungal, insect, vertebrate (such as mammalian cells), and plant cells, including primary cells and immortal cell lines. Numerous representatives of each cell type are commonly used and are available from a wide variety of commercial sources, including, for example, the American Tissue Culture Collection (ATCC). Further details of some specific embodiments are discussed below.
Prokaryotes, such as bacteria, may be used as host cells. Prokaryotic expression systems are advantageous, at least, because of culture affordability, ease of genetic manipulation, and high yields of desired product(s). As described herein, E. coli BL21 (DE3) is a suitable prokaryotic host cell. Other suitable E. coli host cells include, without limitation, E. coli K12 strain 94 (ATCC No. 31,446), coli strain W3 110 (ATCC No, 27,325), E. coli X1776 (ATCC No, 31,537), E. coli B, and many other strains, such as HB101, JM101, NM522, NM538, NM539, B1-21, B1-21 (DE3) pLysS, Origami B, OmpT-defective CD41, CD43 (DE3), and phosphatidylenthanolamine (PE)-deficient AD93. Similarly, other species and genera of prokaryotes including Pseudomonas aeruginosa, Salmonella gastroenteritis (typhimirium), S. typhi, S. enteriditis, Shigella flexneri, S. sonnie, S. dysenteriae, Neisseria gonorrhoeae, N. meningitides, Haemophilus influenzae, H. pleuropneumoniae, Pasteurella haemolytica, P. multilocida, Legionella pneumophila, Treponema pallidum, T. denticola, T. orales, Borrelia burgdorferi, Borrelia spp., Leptospira interrogans, Klebsiella pneumoniae, Proteus vulgaris, P. morganii, P. mirabilis, Rickettsia prowazeki, R. typhi, R. richettsii, Porphyromonas (Bacteriodes) gingivalis, Chlamydia psittaci, C. pneumoniae, C. trachomatis, Campylobacter jejuni, C. intermedis, C. fetus, Helicobacter pylori, Francisella tularenisis, Vibrio cholerae, Vibrio parahaemolyticus, Bordetella pertussis, Burkholderie pseudomallei, Brucella abortus, B. susi, B. melitensis, B. canis, Spirillum minus, Pseudomonas mallei, Aeromonas hydrophila, A. salmonicida, Lactococcus lactis, and Yersinia pestis, may all be used as prokaryotic expression hosts.
Prokaryotic host cells or other host cells with rigid cell walls may be transformed using any method known in the art, including, for example, calcium phosphate precipitation, or electroporation. Representative prokaryote transformation techniques are described in Hanahan et al., “Plasmid Transformation of Escherichia coli and Other Bacteria,”Meth. Enzymol., 204:63-113 (1991), which is hereby incorporated by reference in its entirety.
Vectors typically used for transformation of E. coli include, without limitation, pBR322, pUC18, pUC19, pUC118, pUC119, Bluescript M13 and derivatives thereof. Numerous such plasmids are commercially available and are well known in the art.
Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation). Transcription of DNA is dependent upon the presence of a promoter, which is a DNA sequence that directs the binding of RNA polymerase, and thereby promotes mRNA synthesis. Promoters vary in their strength (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters to obtain a high level of transcription and, hence, expression. Therefore, depending upon the host system utilized, any one of a number of suitable promoters may also be incorporated into the expression vector carrying the nucleic acid molecule(s) of the present invention. For instance, when using E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals, which differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference in its entirety.
Recombinant heme-binding proteins can also be produced in archaeal expression systems. Species that are suitable for serving as hosts include, without limitation, Methanosarcina acetivorans and Sulfolobus solfataricus (see e.g., Albers et al., “Production of Recombinant and Tagged Proteins in the Hyerthermophilic Archaeon Sulfolobus solfataricus,” Appl. Environ. Microbial. 72(1):102-11 (2006) and Jonuscheit et al., “A Reporter Gene System for the Hyperthermophilic Archaean Sulfolobus solfataricus based on a Selectable and Integrative Shuttle Vector,”Mol. Microbiol. 48(5):1241-52 (2003), which is hereby incorporated by reference in its entirety).
Fungal protein expression systems can also be utilized in the methods of the present invention to efficiently produce a functional recombinant heme-binding protein. Fungal species are considered safer than animal cells because they pose little risk of contamination by viruses, prions, or endotoxins. Additionally, fungal systems are more efficient and economical than mammalian expression systems. There are several fungal species which have been used as hosts for the expression of recombinant proteins, including, without limitation, yeast (e.g., Pichia pastoris, Kluyveromyces lactis, and Saccharomyces cerevisiae), soil fungus (e.g., Trichoderma reesei), and black mould fungus (e.g., Aspergillus niger).
Yeast strains and yeast-derived vectors are used commonly for the expression of heterologous proteins. For instance, Pichia pastoris expression systems, may be used to co-express a recombinant heme-binding protein of interest with ferrochelatase (see e.g., Ballew N., “Revolutionizing Protein Production in Fungi,” Innovations in Pharmaceutical Technology 70-76 (June 2004), which is hereby incorporated by reference in its entirety). Such systems include suitable Pichia pastoris strains, vectors, reagents, transformants, sequencing primers, and media. Available strains include KM71H (a prototrophic strain), SMD1168H (a prototrophic strain), and SMD1168 (a pep4 mutant strain) (Invitrogen).
Saccharomyces cerevisiae is also commonly used in heterologous expression systems. The plasmid YRp7 is commonly used as an expression vector in Saccharomyces (Stinchcomb et al., “Isolation and Characterization of a Yeast Chromosomal Replicator,” Nature 282:39-43 (1979); Kingsman et al., “Replication in Saccharomyces Cerevisiae of Plasmid pBR313 Carrying DNA from the Yeast trp1 Region,” Gene 7:141-152 (1979); and Tschemper et al., “Sequence of a Yeast DNA Fragment Containing a Chromosomal Replicator and the TRP1 Gene,” Gene 10:157-166 (1980), which are hereby incorporated by reference in their entirety). This plasmid contains the trp1 gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, such as strains ATCC No. 44,076 and PEP4-1 (Jones, E. W., “Proteinase Mutants of Saccharomyces cerevisiae,” Genetics 85:23-33 (1977), which is hereby incorporated by reference in its entirety). The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Yeast host cells can be transformed using the polyethylene glycol method, as described by Hinnen (Hinnen et al., “Transformation of Yeast,” Proc. Natl. Acad. Sci. U.S.A. 75:1929-1933 (1978), which is hereby incorporated by reference in its entirety). Additional yeast transformation protocols are set forth in Gietz et al., “Improved Method for High Efficiency Transformation of Intact Yeast Cells,” Nucl. Acids Res. 20(6):1425 (1992)) and Reeves et al. “A Yeast Intron as a Translation Terminator in a Plasmid Shuttle Vector,” Yeast Res. 4(6):573-597 (2004), which are hereby incorporated by reference in their entirety.
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., “Isolation and Characterization of the Yeast 3-Phosphoglycerokinase Gene (PGK) by an Immunological Screening Technique,” J. Biol. Chem. 255:12073-12080 (1980), which is hereby incorporated by reference in its entirety) or other glycolytic enzymes (Hess et al., “Cooperation of Glycolytic Enzymes,” J. Adv. Enzyme Reg. 7:149-167 (1969) and Holland et al., “Isolation and Identification of Yeast Messenger Ribonucleic Acids Coding for Enolase, Glyceraldehyde-3-Phosphate Dehydrogenase, and Phosphoglycerate Kinase,” Biochem. 17:4900-4907 (1978), which are hereby incorporated by referenced in their entirety), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In the construction of suitable expression vectors, the termination sequences associated with these genes are also ligated into the expression vector 3′ of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.
Another representative eukaryotic expression system involves the recombinant baculoviruses, Autographa californica nuclear polyhedrosis virus (AcNPV; Summers and Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures Texas Agriculture Experiment Station. Bulletin No. 1555 (1987) and Luckow et al., “Trends in the Development of Baculovirus Expression Vectors,” Biotechnol. 6:47-55 (1987), which are hereby incorporated by referenced in their entirety) and Spodoptera frugiperda. Baculoviruses do not infect humans and can therefore be safely handled in large quantities.
A baculovirus expression vector is prepared as previously described using standard molecular biology techniques. The vector may comprise the polyhedron gene promoter region of a baculovirus, the baculovirus flanking sequences necessary for proper crossover during recombination (the flanking sequences comprise about 200-300 base pairs adjacent to the promoter sequence) and a bacterial origin of replication which permits the construct to replicate in bacteria. In particular examples, the vector is constructed so that a heme-binding protein and ferrochelatase nucleic acid sequences are operably linked to the polyhedron gene promoter (collectively, the “expression construct”) and the expression construct is flanked by the above-described baculovirus flanking sequences. Appropriate transfer vectors compatible with insect host cells are known in the art and include, without limitation, pVL1392, pVL1393, pAcGP67 and pAcSecG2T, which incorporate asecretory signal fused to the desired protein, and pAcGHLT and pAcHLT, which contain GST and 6×His tags (BD Biosciences, Franklin Lakes, N.J.).
Insect host cells (e.g., Spodoptera frugiperda cells) are infected with a recombinant baculovirus and cultured under conditions allowing expression of the baculovirus-encoded heme-binding proteins and ferrochelatase protein or polypeptide. When using insect cells, suitable baculovirus promoters include late promoters, such as 39K protein promoter or basic protein promoter, and very late promoters, such as the p10 and polyhedron promoters. In some cases it may be desirable to use transfer vectors containing multiple baculoviral promoters. The expressed heme-binding protein may be extracted from the insect cells using methods known in the art.
Mammalian host cells may also be used for heterologous expression of a heme-binding protein and ferrochelatase protein or polypeptide. Examples of suitable mammalian cell lines include, without limitation, monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line 293S (Graham et al., “Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5,” J. Gen. Virol. 36:59-74 (1977), which is hereby incorporated by reference in its entirety); baby hamster kidney cells (BHK, ATCC CCL-10); Chinese hamster ovary cells (Urlab et al., “Isolation of Chinese Hamster Cell Mutants Deficient in Dihydrofolate Reductase Activity.” Proc. Natl. Acad. Sci. U.S.A. 77:4216-4220 (1980), which is hereby incorporated by reference in its entirety); mouse sertoli cells (TM4; Mather J. P., “Establishment and Characterization of Two Distinct Mouse Testicular Epithelial Cell Lines,” Biol. Reprod. 23:243-252 (1980), which is hereby incorporated by reference in its entirety); monkey kidney cells (CVI-76, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); human lung cells (W138, ATCC CCL-75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); rat hepatoma cells (HTC, MI.54; Baumann ct al., “Dexamethasone Regulates the Program of Secretory Glycoprotein Synthesis in Hepatoma Tissue Culture Cells,”J. Cell Biol. 85:1-8 (1980), which is hereby incorporated by reference in its entirety); and TRI cells (Mather et al., “Culture of Testicular Cells in Hormone-Supplemented Serum-Free Medium,” Annals N.Y. Acad. Sci. 383:44-68 (1982), which is hereby incorporated by reference in its entirety). Expression vectors for these cells ordinarily include (if necessary) DNA sequences for an origin of replication, a promoter located 5′ of the nucleic acid sequence to be expressed, a ribosome binding site, an RNA splice site, a polyadenylation site, and/or a transcription terminator site.
Promoters used in mammalian expression vectors can be of viral origin. Such viral promoters may be derived from polyoma virus, adenovirus 2, and simian virus 40 (SV40). The SV40 virus contains two promoters that are termed the early and late promoters. These promoters are useful because they are both easily obtained from the virus as one nucleic acid fragment that also contains the viral origin of replication (Fiers et al., “Complete Nucleotide Sequence of SV40 DNA,” Nature 273:113-120 (1978), which is hereby incorporated by reference in its entirety). Smaller or larger SV40 DNA fragments may also be used, provided they contain the approximately 250-bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication. Alternatively, promoters that are naturally associated with the foreign gene (homologous promoters) may be used provided that they are compatible with the host cell line selected for transformation.
An origin of replication may be obtained from an exogenous source, such as SV40 or other virus (e.g., polyoma virus, adenovirus, VSV, BPV) and inserted into the expression vector. Alternatively, the origin of replication may be provided by the host cell chromosomal replication mechanism.
Cell-free translation systems are known in the art, and can be used to synthesize heme-binding proteins using the methods of the present invention (see e.g., Kurland, “Translational Accuracy In vitro,” Cell 28:201-202 (1982) and Pavlov et al., “Rate of Translation of Natural mRNAs in an Optimized In vitro System,”Arch. Biochem. Biophys. 328:9-16 (1996); and Cell-Free Translation Systems, Spirin A. S., ed. (2002); Cell-Free Protein Expression, Swartz J. A., ed. (2003), which are hereby incorporated by reference in their entirety). The most frequently used cell-free translation systems consist of extracts from rabbit reticulocytes, wheat germ and E. coli. All are prepared as crude extracts containing all the macromolecular components (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNA. Each extract is supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. coli lysate), and other co-factors (Mg2+, K+, etc.) that facilitate the function of the particular translation machinery.
Either DNA or RNA, in plasmid or linear form, can be used as the starting material for cell-free protein synthesis. However, DNA starting material is necessarily transcribed to RNA using a “coupled” or “linked” system. A “linked” system generally involves DNA transcription with a bacteriophage polymerase followed by translation in the rabbit reticulocyte lysate or wheat germ lysate. Unlike eukaryotic systems (such as, rabbit reticulocyte or wheat germ) where transcription and translation occur sequentially, transcription and translation occur simultaneously in E. coli cell free systems. Thus. E. coli translation systems are “coupled” and can be performed in the same tube using either a DNA or RNA template. Methods of using E. coli cell-free systems have been described in detail (see e.g., Kigawa et al., “Cell-Free Production and Stable-Isotope Labeling of Milligram Quantities of Proteins,” FEBS Lett. 442:15-19 (1999); Noren et al., “A General Method for Site-Specific Incorporation of Unnatural Amino Acids into Proteins,” Science 244:182-188 (1989); Hanes et al., “In vitro Selection and Evolution of Functional Proteins by Using Ribosome Display,” Proc. Natl. Acad. Sci. U.S.A. 94:4937-4942 (1997); Wilson et al., “The Use of mRNA Display to Select High-Affinity Protein-Binding Peptides,” Proc. Natl. Acad. Sci. U.S.A. 98:3750-3755 (2001); and Sawasaki et al., “A Cell-Free Protein Synthesis System for High-Throughput Proteomics,” Proc. Natl. Acad. Sci. U.S.A. 99(23):14652-14657 (2002), which are hereby incorporated by reference in their entirety). In the E. coli system, it may be advantageous to place a Shine-Dalgarno ribosome binding site upstream of the initiator codon in a DNA template. In particular examples, an E. coli S30 extract system allows expression from DNA vectors containing natural E. coli promoter sequences (such as lac or tac).
Another aspect of the present invention relates to a system for producing functional heme-binding proteins. This system comprises an expression system and one or more expression constructs encoding a recombinant heme-binding protein and a recombinant ferrochelatase.
Suitable expression systems are described above, including cell expression systems (e.g., bacterial, fungal, archaeal, and mammalian) and cell-free expression systems.
The expression constructs of the system will depend on the type of expression system. Suitable expression constructs are described supra. The expression constructs can be in linear form or contained in a plasmid or viral vector, and can further contain regulatory elements, such as a promoter sequence, a ribosome binding sequence, and a nucleic acid molecule encoding a termination sequence, to optimize protein expression. The system of the present invention can include one expression construct encoding both the recombinant heme-binding protein and ferrochelatase. Alternatively, the system can include a first expression construct encoding the recombinant heme-binding protein and a second expression construct encoding the recombinant ferrochelatase.
In accordance with this aspect of the invention, the system for producing functional heme-binding proteins further includes one or more heme precursors. Suitable precursors include δ-amino levulinic acid, succinyl CoA, glycine, glutamate, glutamate-1-semialdehyde, porphobilinogen, hydroxymethylbilane and protoporphyrin as described supra.
Another aspect of the present invention relates to a purified preparation of recombinant functional heme-binding protein.
In a preferred embodiment of the present invention, the purified preparation of functional recombinant heme-binding protein is prepared in accordance with the methods described supra. The purified preparation of recombinant heme-binding protein of the present invention has full heme incorporation and does not contain metal-free porphyrin. The purity of the preparation is assessed by the presence or absence of un-metallated heme, which can be measured by fluorescence spectroscopy and resonance Raman spectroscopy. In a preferred embodiment of the invention, the purified preparation of functional, recombinant heme-binding protein of the present invention is not fluorescent when excited at a wavelength of 397 nm and has a resonance Raman spectrum showing no evidence of free-base porphyrin incorporation (see Examples infra).
Purified preparations of heme-binding proteins of the present invention have a variety of therapeutic, research, and commercial utilities. With regard to therapeutic applications, purified preparations of hemoglobin are desired. Hemoglobin is a heme-binding protein responsible for carrying and delivering oxygen to tissues and organs in animals. Recombinant hemoglobin preparations are used as effective and safe oxygen carriers as an alternative to blood transfusion. A purified preparation of recombinant hemoglobin prepared in accordance with the methods of the present invention is fully functional due to full heme incorporation. Accordingly, the incorporation of a purified preparation of hemoglobin of the present invention into oxygen carrier and blood substitute technologies would improve the oxygen carrying capacity. Suitable oxygen carrier and blood substitute technologies include, without limitation, those disclosed in U.S. Pat. No. 4,412,989 to Iwashits et al., U.S. Pat. No. 6,022,849 to Olsen et al, U.S. Pat. No. 7,329,641 to Fronticellie et al., and U.S. Patent Publication No. 2006/0088583 to Takeoka et al., which are hereby incorporated by reference in their entirety.
Cytochrome P450s are a superfamily of enzymes that are critical to human drug metabolism. These proteins have been implicated in many clinical cases of adverse drug reaction and toxicity stemming from mechanism-based enzyme inhibition and drug-drug interactions. High-throughput assays that identify molecules that inhibit or induce CYP450 early in the drug development process are invaluable for guiding the elimination of candidate drugs that have unwanted metabolic properties and facilitating the production of better clinical candidates. Purified preparations of cytochrome P450 proteins of the present invention will benefit the variety of well established preclinical screening assays that are designed to predict drug metabolism and toxicity (see e.g., Trubetskoy et al., “Highly Minaturized Formats for In Vitro Drug Metabolism Assays Using Vivid Fluorescent Substrate and Recombinant Human Cytochrome P450 Enzymes,” J Bimolecular Screening 56-66 (2005); Donato et al., “Fluorescence-Based Assays for Screening Nine Cytochrome P450 (P450) Activities in Intact Cells Expression Individual Human P450 Enzymes,” Drug Metab. Disposition 32(7):699-706 (2004); Zhang et al., “Cytochrome P450 Reaction-Phenotyping: An Industrial Perspective,” Expert Opin, Drug Metab. Toxicol. 3(5):667-87 (2007); Buters et al., “A Highly Sensitive Tool for the Assay of Cytochrome P450 Enzyme Activity in Rat, Dog, and Man: Direct Fluorescence Monitoring of the Deethylation of 7-ethoxy-4-trifluoromethylcoumarin,” Biochem. Pharm. 46(20):1577-1584 (1993); and Yim et al., “A Continuous Spectrophotometric Assay for NADPH-cytochrome P450 Reductase Activity Using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide,” J. Biochem. Mol. Biol. 38(3):366-369 (2005), which are hereby incorporated by reference in their entirety).
Purified preparations of nitric oxide synthases of the present invention also have therapeutic utility with regard to the treatment of vascular diseases, cancer, microbial infections, tissue injury, and neurological pathologies, and for promoting wound healing (see e.g., U.S. Pat. No. 5,658,565 to Billiar et al., U.S. Patent Publication No. 20070071725 to Paterson et al., and U.S. Patent Publication No. 20100087370 to Jain et al., which are hereby incorporated by reference in their entirety).
Purified preparations of cyclooxygenases (e.g. COX-1, COX-2, COX-3) of the present invention also have therapeutic/commercial utility. These enzymes mediate the formation of prostanoids, including prostaglandins, prostacyclin, and thromboxane. Pharmacological inhibition of these enzymes provides relief from the symptoms of inflammation and pain. Accordingly, purified preparations of cyclooxygenases made in accordance with the present invention can be used to screen and identify, using methods described infra, more potent inhibitors than the currently available non-steroidal anti-inflammatory drugs.
In addition to therapeutic utility, purified preparations of heme-binding proteins also have biotechnological utilities. For example, lignin peroxidase is a heme-binding protein that degrades plant lignin, a heterogenous aromatic polymer that encases the cellulose fibers of lignocellulose (see Weng et al., “Emerging Strategies in Lignin Engineering and Degradation for Cellulosic Biofuel Production,” Curr. Opin. Biotech. 19:166-172 (2008), which is hereby incorporated by reference in its entirety). The lignocellulose content of plant biomass is a primary source of renewable carbon that can be used to produce bio-ethanol and chemical feedstocks for commercial use. A major obstacle to exploiting this renewable carbon source is the presence and degradation of lignin. Therefore, lignin degradation via lignin peroxidase, offers an attractive strategy for optimizing biofuel production (see e.g., U.S Patent Application Publication Nos. 2010/0291653 to Ness et al., 2010/0017916 to Pappan et al., 2005/0233423 to Berka et al., which are hereby incorporated by reference in their entirety). Accordingly, the present invention contemplates the use of a purified preparation of lignin peroxidase that does not contain metal-free porphyrin, for biofuel production.
Another aspect of the present invention relates to a method of identifying an agent that modulates activity of a heme-binding protein. This method involves providing a candidate agent and providing a recombinant functional heme-binding protein. This method further involves contacting the candidate agent with the recombinant functional heme-binding protein under conditions at which the functional heme-binding protein is active and comparing the activity of the functional heme-binding protein as a result of said contacting to the activity of the heme-binding protein alone, both under said conditions at which the heme-binding protein is active. A candidate agent that modulates the activity of a heme-binding protein is identified based on said comparing.
A related aspect of the present invention relates to a method of evaluating the metabolism of an agent by a heme-binding protein. This method involves providing a candidate agent and providing a recombinant functional heme-binding protein. This method further involves contacting the candidate agent with the recombinant functional heme-binding protein under conditions at which the functional heme-binding protein is active and comparing the activity of the functional heme-binding protein as a result of said contacting to the activity of the heme-binding protein alone, both under said conditions at which the heme-binding protein is active. The metabolism of the candidate agent by a heme-binding protein is evaluated based on said comparing.
In one embodiment of this aspect of the present invention, the above methods further involve providing a heme-binding protein substrate and/or one or more heme-binding protein co-factors, where the heme-binding protein substrate and/or heme-binding co-factor is present when the heme-binding protein is contacted with the candidate agent. The activity of the heme-binding protein in the presence of the one or more heme-binding cofactors with the heme-binding protein substrate is evaluated. In other words, the rate of metabolism or conversion of the heme-binding protein substrate by the heme-binding protein is a measure of heme-binding protein activity.
In accordance with this aspect of the present invention a decrease in the activity of the functional heme-binding protein in the presence of the candidate agent compared to in the absence of the candidate agent identifies an agent that modulates the heme-binding protein activity. In this regard, the candidate agent is an inhibitor of heme-binding protein activity. Activators of heme-binding protein activity can also be identified using this assay. An increase in heme-binding protein activity would identify candidate activators of heme-binding protein activity.
In accordance with aspects of the present invention directed to evaluating the metabolism of an agent by a heme-binding protein, an increase in the activity of a functional heme-binding protein in the presence of the candidate agent compared to in the absence of the candidate agent may identify an agent that is a substrate for heme-binding protein metabolism. Alternatively, a decrease in heme-binding protein activity may also indicate that the candidate agent is a substrate for the heme-binding protein activity (e.g., a substrate for cytochrome P450 metabolism). In this case, the observed decrease in heme-binding protein activity would result from competition for heme-protein binding between the heme-binding protein substrate and the candidate agent. In either case, metabolism of the candidate agent by the heme-binding protein can be further evaluated by analyzing the metabolic profile of the candidate agent using methods known in the art (e.g., high-performance liquid chromatography).
In accordance with these aspects of the present invention, the recombinant functional heme-binding protein does not contain metal-free porphyrin. Any of the heme-binding proteins described supra can be utilized in this aspect of the invention. In one embodiment of the present invention, the heme-binding protein is a cytochrome P450 protein. As noted above, the cytochrome P450 family of enzymes are the major catalysts for the oxidative metabolism of a vast array of hydrophobic chemicals. These enzymes are involved in the metabolism or biotransformation of endogenous as well as exogenous hydrophobic compounds. Since cytochrome P450-mediated metabolism influences drug clearance, toxicity, activation, and in some cases, adverse interaction with other drugs, accurately identifying agents that modulate cytochrome P450 activity or serve as substrates for metabolism is particularly important for early toxicological screening of candidate drugs. A number of cytochrome P450 screening assays are known in the art (see e.g., Donato et al., “Fluorescence-Based Assays for Screening Nine Cytochrome P450 (P450) Activities in Intact Cells Expression Individual Human P450 Enzymes,” Drug Metab. Disposition 32(7):699-706 (2004); Zhang et al., “Cytochrome P450 Reaction-Phenotyping: An Industrial Perspective,” Expert Opin. Drug Metab. Toxicol, 3(5):667-87 (2007); Buters et al., “A Highly Sensitive Tool for the Assay of Cytochrome P450 Enzyme Activity in Rat, Dog, and Man: Direct Fluorescence Monitoring of the Deethylation of 7-ethoxy-4-trifluoromethylcoumarin,” Biochem, Pharm. 46(20):1577-1584 (1993); and Yim et al., “A Continuous Spectrophotometric Assay for NADPH-cytochrome P450 Reductase Activity Using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide,” J. Biochem. Mol. Biol. 38(3066-369 (2005), which are hereby incorporated by reference in their entirety) and are commercially available, especially in high-throughput formats (see e.g. Promega. Invitrogen, and Agilent Technologies), all of which can be utilized in these aspects of the present invention, Incorporation of purified preparations of recombinant cytochrome P450 proteins of the present invention will enhance the accuracy of these and other P450 enzyme assays known and used in academic and pharmaceutical research arenas.
The following examples illustrate various methods for compositions in the treatment method of the invention. The examples are intended to illustrate, but in no way limit, the scope of the invention.
Co-expression of Ferrochelatase with gsNOS. Ferrochelatase (FC) and gsNOS were expressed from the same pACYCduet vector (Novagen). To clone FC, genomic DNA was extracted from E. coli BL21(DE3) cells with the genomic DNA extraction kit from Epicenter. The FC gene was then PCR-amplified (Phusion polymerase, New England Biolabs) from the genomic DNA with primers that generated Nde1 and Xho1 sites at the 5′ and 3′ ends of the gene, respectively. A stop codon was introduced into the 3′ primer before the Xho1 site to prevent C-terminal attachment of the vector-supplied S-tag. The amplified FC gene was then cloned into the Nde1 and Xho1 sites in Multiple Cloning Site-2 of the pACYCdtiet vector. The gene for gsNOS was derived from a previous pET28a-gsNOS plasmid (Sudhamsu et al., “Structure and Reactivity of a Thermostable Prokaryotic Nitric-Oxide Synthase that Forms a Long-Lived Oxy-Heme Complex,” J. Biol. Chem. 281:9623-9632 (2006), which is hereby incorporated by reference in its entirety) by digesting the vector with Nco1 and Xho1 so as to include the His-tag and the thrombin cleavage site along with the coding sequence for gsNOS in the excised fragment. The Nco1-Xho1 fragment was then cloned into the pACYCduet-FC plasmid between the Nco1 and Sal1 sites. Sal1 and Xho1 produce compatible cohesive ends and thereby allow the His-tag, thrombin cleavage site and gsNOS fragment to be cloned between the Nco1 and Sal1 sites of Multiple cloning site-1 of the pACYCduct-FC plasmid. The resulting pACYCduet plasmid allows over-expression of gsNOS with a cleavable His-tag and FC with no tag, GsNOS was expressed and purified as reported before (Sudhamsu et al., “Structure and Reactivity of a Thermostable Prokaryotic Nitric-Oxide Synthase that Forms a Long-Lived Oxy-Heme Complex,” J. Biol. Chem. 281:9623-9632 (2006), which is hereby incorporated by reference in its entirety). Co-expression of gsNOS and FC was also performed similarly to expression of gsNOS alone, although, a lesser amount of δ-ALA was added at the time of induction (10 mg/L versus 25 mg/L for gsNOS), and the growth media was supplemented with 100 μM FeCl3. The antibiotics, chloramphenicol (34 μg/L) and kanamycin (50 μg/L), were added to the growth media of pACYCduet-gsNOS-FC and pET28a-gsNOS plasmids, respectively.
Co-expression of Ferrochelatase with BP450 and HBPAS. For co-expression of BP450 and HBPAS the same procedure was used. BP450 (NCBI: CBG70284) was cloned into pET151/D-TOPO (Invitrogen), a directional cloning vector with an N-terminal 6×His-Tag followed by a TEV cleavage site and an ampicillin selectable marker. HBPAS (NCBI: NP—248866) was cloned into pET28a (Novagen) using NdeI and HindIII restriction sites, which included an N-terminal 6×His-Tag followed by a thrombin cleavage site and a kanamycin selectable maker. Competent E. coli BL21 (DE3) cells containing FC/pACYCduet were transformed with either BP450/pET151/D-TOPO or HBPAS/pET28. Cells were grown at 37° C. in Luria broth containing 20 ug/mL Cm and 100 ug/L Amp (BP450) or 50 ug/L Kan (HBPAS) to an OD=0.6-0.8, Prior to induction with IPTG, the temperature was reduced to 17° C. and 25 mg/L d-ALA was added to the growth media. Cells were harvested 18-20 hrs after induction. An identical procedure with cells lacking the FC plasmid was used to express BP450 and HBPAS without FC. Both proteins were purified using Ni-NTA (Qiagen) chromatography techniques following the manufacturer's protocol. Furthermore, the proteins were purified to >95% purity using size exclusion chromatography after the removal of 6×His.
Spectroscopy. Resonance Raman and UV-Visible spectra were recorded as described previously (Kabir et al., “Substrate-Ligand Interactions in Geobacillus Stearothermophilus Nitric Oxide Synthase,” Biochem. 47:12389-12397 (2008), which is hereby incorporated by reference in its entirety).
Materials. Sodium chloride was obtained from Mallinkrodt, Ferric Chloride, IPTG and TRIS were from Fisher Scientific, Kanamycin, and Chloramphenicol from USBiological. δ-ALA was obtained from Sigma-Aldrich.
GsNOS (Geobacillus stearothermophihis Nitric Oxide Synthase) is a thermophilic enzyme that forms a highly stable heme-oxygen complex (Sudhamsu et al., “Structure and Reactivity of a Thermostable Prokaryotic Nitric-Oxide Synthase that Forms a Long-Lived Oxy-Heme Complex,”J. Biol. Chem. 281:9623-9632 (2006), which is hereby incorporated by reference in its entirety) that has helped in identification of catalytic intermediates responsible for L-arginine oxidation to nitric oxide (Davydov et al., “EPR And ENDOR Characterization of the Reactive Intermediates in the Generation of NO by Cryoreduced Oxy-Nitric Oxide Synthase from G. Stearotherniophilus,”J. Am. Chem. Soc. 131:14493-14507 (2009), which is hereby incorporated by reference in its entirety). In the over-expression of heme proteins the heme precursor δ-ALA is routinely added to the growth media when protein production is induced. Such δ-ALA supplementation results in complete heme incorporation for two other bacterial NOS proteins: B. subtilis NOS (Pant et al., “Structure of a Nitric Oxide Synthase Heme Protein from Bacillus Subtilis,” Biochem. 41:11071-11079 (2002), which is hereby incorporated by reference in its entirety) and D. radiodurans NOS (Buddha et al., “Regioselective Nitration of Tryptophan by a Complex Between Bacterial Nitric-Oxide Synthase and Tryptophanyl-Trna Synthetase,” J. Biol. Chem. 279:49567-49570 (2004), which is hereby incorporated by reference in its entirety). However, in what follows, it is shown that gsNOS over-expressed and purified from E. coli consists of two species: native heme-containing gsNOS, and gsNOS with protoporphyrin IX (free-base porphyrin) bound instead of heme. It was hypothesized that co-expression of ferrochelatase, the enzyme that metallates porphyrin would ameliorate this problem.
A UV-Vis spectroscopic analysis of gsNOS over-expressed in E. coli shows that the amount of heme incorporated with the protein changes from batch to batch, with the ratio of Soret peak height (403 nm) to protein peak height (280 nm) (Abs403/Abs280) varying between 0.25-0.40. Co-expression of FC with gsNOS increased Abs403/Abs280 to 0.6 (
Purified gsNOS, when over-expressed in E. coli with 25 mg/L δ-ALA added at the time of induction, results in two bands of −42 kDa on SDS-PAGE (
Confirmation of porphyrin incorporation into gsNOS came from resonance Raman studies of gsNOS in the presence of substrate L-arginine. A sample of gsNOS (expressed without FC) in the presence of substrate L-arginine, shows vibrational frequencies at 662 cm−1, 738 cm−1, 783 cm−1, 1360 cm−1 and 1543 cm−1, apart from the typical vibrational frequencies that have been previously observed (Santolini et al., “Resonance Raman Study of Bacillus Subtilis NO Synthase-Like Protein: Similarities and Differences with Mammalian NO Synthases,” Biochem. 45:1480-1489 (2006) and Rousseau et al., “Ligand-Protein Interactions in Nitric Oxide Synthase,” J. Inorganic Biochem. 99:306-323 (2005), which are hereby incorporated by reference in their entirety) for other NOSs (
Whether foregoing the addition of δ-ALA was also tested, while co-expressing FC would result in complete heme incorporation of gsNOS. The purified protein, which was checked for heme content by both SDS-PAGE and by UV-Vis spectroscopy had a higher degree of home incorporation than under conditions of only adding δ-ALA (heme:protein ratio ˜0.5); however, heme incorporation was not complete, Addition of a small amount of δ-ALA, (10 mg/L) is sufficient to make up for the slow rate of δ-ALA biosynthesis and produce fully incorporated protein in the presence of FC.
In addition to gsNOS, FC also increases heme content to saturating levels in two other unrelated proteins: BP450, a Cys-ligated heme protein and HBPAS: a His-ligated heme protein. Both of these proteins, when over-expressed in E. coli, are produced with partial heme incorporation, UV-Vis spectra of purified BP450 and BP450 co-expressed with FC are strikingly different, with the increased intensity of the Soret peak indicative of greater heme content in the material produced along with FC (
The production of δ-ALA is a rate-limiting step for heme biosynthesis (Ades, I. Z., “Heme Production in Animal-Tissues—The Regulation of Biogenesis of Delta-Aminolevulinate Synthase,” Internat. Biochem. 22:565-578 (1990); Woodard et al., “Regulation of Heine-Biosynthesis in Escherichia-Coli,” Archives Biochem. Biophys. 316:110-115 (1995); Gibson et al., “Is Delta-Aminolevulinic Acid Dehydratase Rate Limiting in Heme Biosynthesis Following Exposure of Cells to Delta-Aminolevulinic Acid?” Photochem. Photobiol. 73:312-317 (2001); and Heinemann et al., “The Biochemistry of Heme Biosynthesis,” Archives Biochem. Biophys. 474:238-251 (2008), which are hereby incorporated by reference in their entirety) and δ-ALA synthesis is itself slowed by heme feedback inhibition. Thus, as has been well recognized (Ades, I. Z., “Heme Production in Animal-Tissues—The Regulation of Biogenesis of Delta-Aminolevulinate Synthase,”Internat. J. Biochem. 22:565-578 (1990); Woodard et al., “Regulation of Heme-Biosynthesis in Escherichia-Coli,” Archives Biochem. Biophys. 316:110-115 (1995); Gibson et al., “Is Delta-Aminolevulinic Acid Dehydratase Rate Limiting in Heme Biosynthesis Following Exposure of Cells to Delta-Aminolevulinic Acid?” Photochem. Photobtol. 73:312-317 (2001); and Heinemann et al., “The Biochemistry of Heme Biosynthesis,”Archives Biochem. Biophys. 474:238-251 (2008), which are hereby incorporated by reference in their entirety), feeding with δ-ALA greatly aids recombinant heme protein production in E. coli. However, as demonstrated herein, under conditions of augmentation with δ-ALA, ferrous iron insertion into protoporphyrin IX becomes rate-limiting. Co-expression with ferrochelatase along with the addition of a small amount of δ-ALA, is sufficient to produce fully incorporated heme protein. This method is applicable for both Cys-ligated and His-ligated heme proteins. In the case of the two Cys-ligated proteins, porphyrin substitution could be observed on an SDS-PAGE gel as two closely spaced bands and also by fluorescence spectroscopy. In the one His-ligated heme protein example, UV-Visible and fluorescence spectra were effective indicators of insufficient porphyrin metallation, but only one band was observed by SDS-PAGE even with less than full porphyrin content. This is probably because the heme or porphyrin does not remain associated with the PAS protein during electrophoresis, unlike the other two cases.
In conclusion, the simple and inexpensive method of co-expressing ferrochelatase is effective at producing fully incorporated heme proteins in E. coli.
Co-expression of FC and full-length NOS was carried out as described supra. Under normal conditions of expression the Soret band indicative of heme incorporation is barely visible above the flavin absorption of the reductase component (gray trace), however, upon co-expression with ferrochelatase a strong Soret absorption peak (at ˜416 nm), indicative of heme incorporation becomes apparent (black trace). Total yields of full-length NOS also increase in the presence of ferrochelatase as indicated by the overall greater cofactor absorption profile.
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/347,193, filed May 21, 2010, which is hereby incorporated by reference in its entirety.
This invention was made with government support under grant number NCHE-0749997 awarded by the National Science Foundation and grant number R01GM079679 awarded by the National Institutes of Health. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61347193 | May 2010 | US |
